Outbreaks of leptospirosis are of significant and growing public health concern in many tropical and subtropical countries. Leptospirosis is a zoonotic disease caused by spirochaetes of the genus Leptospira, which are classified into 9 pathogenic species and more than 200 serovars on the basis of structural heterogeneity in the carbohydrate component of the lipopolysaccharide. Human infections are endemic in most tropical and most moderate climates. Globally, an estimated number of >500,000 severe cases occur annually with fatality rates exceeding 10%.
This neglected infectious disease is also reported to be an emerging or re-emerging disease in industrialized countries, with probable increasing impacts due to global warming and increasing travel-related cases [Lau C, Travel Med Infect Dis. 2010]. The incidence is underestimated due to highly variable clinical presentation which is characterized by non-specific signs and symptoms; leptospirosis is often confused with other diseases such as dengue, rickettsiosis, enteric fevers and malaria. The complete triad of Weil's disease (hepatic failure, renal failure and hemorrhage) is recognized to account for less than one third of human cases [Mac Bride A. J. et al., Curr. Opin. Infect. Dis. 2005]. Most of the early signs and symptoms point to the so-called “acute fever of unknown origin” (FUO), a major diagnostic challenge in tropical and subtropical areas. Because of nonspecific symptoms in human leptospirosis, the biological confirmation is needed to ascertain the disease. In many endemic regions, the laboratory diagnosis of leptospirosis is not available due to a lack of reliable, rapid and simple diagnostic assay for a point of care diagnosis of human leptospirosis.
Yet, an early and proper antibiotic treatment is a key determinant of the outcome in leptospirosis [Suputtamongkol Y. et al, PLoSNegl. Trop. Dis. 2010], because, in contrast to many similar diseases (e.g. dengue), leptospirosis can easily be treated with antibiotics in its early stages. However, the diagnosis has to be confirmed before the 5th day after disease onset, when treatment with antibiotics is most effective.
The need for reliable Rapid Diagnostic Tests (RDTs) for diagnosing human leptospirosis has therefore been largely recognized. Preferably, these RDTs should be portable, so that they can be used directly on the bedside, even in remote health centers, so as to improve clinical management of leptospirosis patients in remote dispensaries of tropical and subtropical regions.
Currently, the laboratory diagnosis for leptospirosis relies on the detection of antibodies raised against the Leptospira bacteria by serological techniques such as Microscopic agglutination test (MAT), or of spirochaete nucleic acids by PCR [Goarant C. Trop. Med. Int. Health, 2009). However, these techniques are inappropriate for early clinical care in peripheral health centers that support the major part of the leptospirosis burden, because they are time-consuming and require sophisticated materials that are most frequently available only in central reference laboratories [Hartskeerl R A, Clin. Microbiol. Infect. 2011]. Also, serological techniques such as Microscopic agglutination test (MAT) are limited by the fact that they use few representative group of serovar antigens, so that a negative reaction on serial samples does not rule out the possibility that the patient might actually be infected with a Leptospira serovar not included in the battery of the tested antigens. PCR-based techniques, although very sensitive and early tests, are technically demanding and the leptospires disappear from the blood vessels approximately at day 7 after infection.
Moreover, ELISA-based assays using crude whole-cell lysates of Leptospira strains (usually the saprophyte L. biflexa serovar Patoc strain Patoc 1) as antigens may not recognize the diversity of circulating strains and the sensitivity of these tests is generally poor (Mc Bride A J. et al, Curr. Opinion Infect Dis, 2005).
Thus, the biological confirmation of leptospirosis is currently not satisfactory, as it is not reliable, tedious and rarely available in a timely manner.
In fact, a major challenge is still to discover antigens that are conserved across the major Leptospira strains, since such antigens would potentially be recognized by most of the antibodies generated in Leptospira-infected patients.
In this context, the present inventors have identified a Leptospira antigen which allows the detection of a broad spectrum of antibodies directed against most of the serovars. This Leptospira antigen is expressed by the bacteria of the serovar Hurstbridge, which was originally isolated from pigs in Australia [Perolat P. et al, Int. J. Syst. Bacteriol. 1998] and is now classified in the species Leptospira fainei, known to also infect humans [Chappel R J. et al, Epidemiol. Infect., 1998]. The present inventors indeed demonstrated that this antigen presents a high reactivity towards antibodies generated by several leptospirosis serogroups, even distantly serologically related, such as serogroups Australis, Autumnalis, Ballum, Bataviae, Canicola, Cynopteri, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae, Panama, Pomona, Pyrogenes, Sejroe and Tarassovi, on serum samples of a number of metropolitan French patients. Of note, false positive results are excluded since the serovar Hurstbridge is poorly represented outside Australia and New Zealand. The present inventors also identified a method to inactivate the bacteria of the serovar Hurstbridge so as to enhance the exposure of said antigen at the bacterial cell surface.
Using such an antigen, it is possible to develop different Rapid Diagnostic Tests (RDTs) exhibiting high sensitivity and specificity for numerous Leptospira serovars.
As disclosed below in detail, the present inventors developed two different RDTs, that are i) an ELISA test and ii) a vertical flow immunochromatography dipstick assay, these two RDTs using heat-inactivated Leptospira fainei bacteria as antigen, for detecting anti-Leptospira human IgM in human serum samples. The robustness of these two RDTs is very satisfactory in terms of sensitivity, specificity, reproducibility, and shelf-life.
It is possible to use the ELISA and dipstick assay of the invention as robust, simple, and rapid diagnostic tools for diagnosing leptospirosis in patients presenting early signs and symptoms thereof.
Moreover, as the dipstick assay of the invention does not require expensive and complex analysis system, it can therefore be used in remote health centers, or in remote dispensaries of tropical and subtropical regions.
As Leptospira fainei bacteria presents a high reactivity towards antibodies generated by several leptospirosis serogroups, the present inventors also propose to use Leptospira fainei bacteria as antigen in a Microscopic Agglutination Test (MAT).